Oncogenic ras-p21 protein, but not its wild-type counterpart protein, induces malignant transformation of mammalian cell lines such as NIH 3T3 cells (1) and has been implicated as a major causative factor in a high proportion of human solid tissue tumors (2). In Xenopus laevis oocytes, microinjection of oncogenic (containing Val in place of Gly 12), but not wild-type, p21 induces oocyte maturation (3). Insulin induces oocyte maturation and requires activation of normal cellular ras-p21 (4).
Several agents that strongly block Val 12-p21-induced oocyte maturation have virtually no effect on insulin-induced maturation (5). Among these agents are specific peptides, identified from molecular modeling studies, that correspond to effector domains from both ras-p21 itself, such as the 35-47, 96-110 and 115-126 sequences (5) and from some of its target proteins such as the ras-binding domain of raf (residues 97-110) (6-8) and the SOS guanine nucleotide exchange protein (residues 994-1004) (9, 10). These peptide domains were identified as those that change conformation in response to the presence of single oncogenic amino acid substitutions at positions 12 or 61 or multiple substitutions at positions 10, 12 and 59 when the computed average structures for these proteins either alone or in complex with target proteins were superimposed on that for the wild-type protein.
The finding that these peptides (in addition to other agents) block oncogenic ras-p21 selectively indicates that the oncogenic protein induces mitogenesis by pathways that may overlap with, but are also distinct from, pathways utilized by the wild-type protein. In studies designed to identify pathway differences, it was found that, in oocytes, oncogenic but not insulin-activated wild-type ras-p21 interacts with the transcriptional activating protein, jun and its kinase, jun kinase (JNK) (11, 12), and requires the presence of protein kinase C (PKC) (13). In these studies, it was determined that the peptide whose sequence corresponds to p21 residues 96-110, called PNC-2, blocks the interaction of Val 12-p21 with JNK (11, 12) in a dose-response curve that superimposes on that for its inhibition of Val 12-p21-induced oocyte maturation (5).
Additionally, the peptide whose sequence corresponds to p21 residues 35-47, called PNC-7, encompasses a domain of the protein implicated in its interacting with multiple targets including raf p74 protein, GTPase activating protein (GAP) and the guanine nucleotide exchange protein, SOS (reviewed in ref. 5). This peptide strongly inhibits c-raf-induced oocyte maturation but has no effect on oocyte maturation induced by an oncogenic mutant raf lacking the ras binding domain (RBD) in its amino terminal regulatory domain (14). Both PNC-2 and 7 appear to act on different steps on the oncogenic ras-p21 signal transduction pathway. For example, PNC-2 but not PNC-7 interferes with Val 12-p21-JNK interaction (11, 12) while PNC-7 but not PNC-2 blocks signal transduction through c-raf (15).
Since various cancers involve expression of Val 12-p21 protein, as well as other oncogenic proteins, it would be useful to be able to inhibit expression of such proteins. For example, pancreatic cancer is a nearly always fatal disease with a median survival time of only 80-90 days for a patient diagnosed with the disease. Pancreatic cancer is one of the more lethal forms of cancer in numbers of patients killed in the U.S. Less than 4% of patients are alive 5 years from the time of diagnosis. The present invention provides peptides and pharmaceutical compositions comprising such peptides which when administered to pancreatic cancer cells, not only inhibit oncogenic Val 12-p21 but actually cause cancerous cells to phenotypically revert to non-cancerous cells. The present invention is therefore useful in treating various types of cancers which express Val 12-p21 protein and/or other oncogenic proteins. Treatment of ras-induced tumors converts malignant masses into benign ones, allowing for the halting of metastatic disease.